1. Field of the Invention
The present invention relates generally to avian vaccines against avian influenza (AI). More specifically, the present invention provides a recombinant turkey herpesvirus modified by the presence of the cDNA encoding the hemagglutinin (HA) protein of avian influenza virus under a promoter.
2. Description of the Related Art
Avian influenza is caused by avian influenza viruses that are classified in the family Orthomyxoviridae, genus Influenzavirus A. The genome of the avian influenza virus consists of eight segments of single-stranded, negative-sense RNA. The viral genome encodes ten proteins, of which eight proteins are structural proteins including HA and neuraminidase (NA), and two proteins are nonstructural. Influenza A viruses are divided into subtypes based on antigenicity of HA and NA proteins. There are 16 HA antigens and nine NA antigens recognized. HA is considered the major antigen that can elicit protective antibodies in birds.
Influenza A viruses from poultry are categorized into two pathotypes based on their pathogenicity: highly pathogenic avian influenza (HPAI) viruses and low pathogenic avian influenza (LPAI) viruses. Most avian influenza viruses are of low virulence, but a few viruses of H5 and H7 subtypes can cause severe systemic disease that results in high mortality. Although only a few H5 and H7 avian influenza viruses are of high virulence, all H5 and H7 viruses are identified as notifiable avian influenza virus by World Organization for Animal Health (OIE) because of the risk of low virulent viruses increasing virulence by mutation.
Since the late 1990s, there has been a significant increase in the number of AI outbreaks and in the number of birds involved in those outbreaks (I. Capua et al., 2004, Avian Pathology, 33: 393-404). The most notable example is a series of H5N1 HPAI outbreaks in China and South-East Asia, which has now spread to other parts of the world such as Europe, the Middle East, and Africa. The outbreaks have cost as many as 160 human lives in more than 10 countries since 2003 although apparent human-to-human transmission has yet to be confirmed. These recent outbreaks have caused tremendous economic losses to the poultry industry and raised public concerns because of fear of a possible human pandemic.
Vaccination against AI had not been conducted extensively until recently because the so-called “stamping-out” procedure has been the primary option. In the “stamping-out” procedure, all chickens in flocks infected with AI are culled. Most AI outbreaks were eradicated or controlled by “stamping-out” in the past. However, in recent AI outbreaks, especially in the H5N1 outbreaks, there have been situations in which massive culling was not practical or feasible due to intolerable economic costs and losses associated with the culling, widespread presence of so-called backyard chickens, and so forth. In those situations, vaccination has been considered a suitable and powerful tool to support AI eradication or AI control programs because vaccination has been shown to protect poultry against clinical signs and death and reduce virus shedding in vaccinated birds, thereby reducing transmission of virus (D. E. Swayne, 2003, Developments in Biologicals, 114: 201-212). In order to utilize vaccines in AI eradication programs or AI control programs, it is critical for trade and surveillance purposes that vaccinated birds may be differentiated from those infected with the field virus. In fact, field exposure in vaccinated flocks must be detected in simple serological assays. Otherwise, the field virus may circulate in the vaccinated birds undetected. It is also important that evidence of vaccination may be detected by simple assays in order to confirm that most or all birds in vaccinated flocks are properly vaccinated.
Commercial vaccines currently available are inactivated whole AI antigens with oil adjuvant and a fowlpox virus vectored recombinant vaccine. Although both vaccines have been shown to be efficacious, they require labor-intensive and expensive parenteral vaccination that involves handling each bird manually. While the inactivated AI vaccines have been used in the program called DIVA (“Differentiation of infected from vaccinated animals”), there have been no commercially available tests developed for mass application. It has been shown that chickens pre-immunized with fowlpox virus either by field exposure or by vaccination with conventional fowlpox vaccines would not develop consistent protective immunity against AI after vaccinated with the fowlpox virus vectored recombinant AI vaccine (D. E. Swayne et al., 2000, Avian Diseases, 44: 132-137). The fowlpox virus vectored recombinant AI vaccine has failed to elicit serological response detectable by the hemagglutination inhibition (HI) test consistently (D. E. Swayne et al., 1997, Avian Diseases, 41: 910-922). Hence, development of vaccines that are easier to administer and that may be readily differentiated from field virus infection is desirable for the poultry industry.
The commercial fowlpox virus vectored recombinant AI vaccine contains the HA gene of the AI virus A/turkey/Ireland/1378/83 (H5N8) (J. R. Taylor et al., 1988, Vaccine, 6: 504-508). Several other experimental fowlpox vectored recombinant vaccines have been developed and shown to be efficacious against challenge with AI viruses in experimental conditions. Avian influenza virus genes contained in the fowlpox vectored recombinant vaccines include the HA gene from A/Chicken/Scotland/59 (H5N1) (C. W. Beard et al., 1991, Avian Diseases, 35: 356-359) and the HA and NA genes from A/Goose/Guangdond/3/96 (H5N1) (C. Qiao et al., 2003, Avian Pathol., 32:25-31). M. Mingxiao et al. fused the HA genes from H5N1 subtype and H7N1 subtype to form a single open frame and inserted into a recombinant fowlpox virus along with chicken Interleukin-18 (M. Mingxiao et al., 2006, Vaccine, 24: 4304-4311). Broad cross protection among the AI virus H5 subtypes has been observed. The fowlpox virus vectored recombinant AI vaccine and the inactivated whole AI vaccines for avian influenza H5 subtypes have been demonstrated to protect chickens against challenge with diverse H5 subtype AI viruses, of which deduced HA amino acid sequence similarities with the vaccines are as low as 87% (D. E. Swayne et al., 2000, Veterinary Microbiol., 74: 165-172).
Next generation vaccines under development include recombinant Newcastle disease virus vaccines (D. E. Swayne et al., 2003, Avian Diseases, 47: 1047-1050; J. Veits et al., 2006, Proc. Natl. Acad. Sci. U.S.A., 103:8197-8202; M. Park et al., 2006, Proc. Natl. Acad. Sci. U.S.A., 103:8203-8208; and J. Ge et al., 2007, J. Virol. 81: 150-158), recombinant infectious laryngotracheitis virus vaccines (D. Luschow et al., 2001, Vaccine 19: 4249-4259 and J. Veits et al., 2003, J. Gen. Virol. 84: 3343-3352), a recombinant adenovirus vaccine (W. Gao et al., 2006, J. Virol. 80: 1959-1964), baculovirus-expressed subunit vaccines (J. Crawford et al., 1999, Vaccine, 17:2265-2274 and D. E. Swayne et al., 2001, Avian Diseases, 45: 355-365) and DNA vaccines (U.S. Pat. No. 5,916,879 and M. Cherbonnel et al., 2003, Avian Diseases, 47: 1181-1186). Although the recombinant Newcastle disease virus vaccines, the recombinant infectious laryngotracheitis virus vaccines, and the recombinant adenovirus vaccine were able to confer partial to semi-complete protection against AI challenge in specific pathogen free chickens, their efficacy in chickens with maternal antibodies to the vector viruses or AI, or in chickens with previous infection or vaccination with the vector viruses remains to be demonstrated. The DNA vaccines have also shown to provide protective immunity in chickens, but they require at least two vaccinations and individual administration to each chicken. The baculovirus-expressed subunit vaccines also require individual administration to each chicken.
Turkey herpesvirus (HVT), Marek's disease virus (MDV) serotype-3, has been used as a vector to express antigens from avian pathogens. Wild type HVT or recombinant HVT can be administered to either the late developmental stage of embryos via the in ovo route or one-day-old chicks via the subcutaneous route at hatcheries. Recombinant, cell-associated HVT vaccines, after inoculation into embryos or one-day-old chicks with maternal antibodies to inserted antigens, are demonstrated to be able to overcome influences of maternal antibodies and confer protective immunity to chickens as maternal antibodies wane (U.S. Pat. No. 6,764,684 and U.S. Pat. No. 6,866,852). Excellent duration of immunity is also achieved by recombinant HVT (U.S. Pat. No. 6,866,852) probably because HVT goes latent and stays inside vaccinated birds for their whole life. Thus, HVT may be considered an excellent vector for avian pathogens. There have been no reports of constructing recombinant HVT or MDV with avian influenza antigens. Although claim 15 of U.S. Pat. No. 5,853,733 describes the recombinant HVT comprising a polypeptide gene of AI virus inserted within a region which corresponds to an EcoRI #9 fragment of the HVT genome, there is no actual example of constructing recombinant HVT with avian influenza antigens. In mammalian species, U.S. Pat. No. 6,225,111 describes construction of recombinant equine herpesviruses containing the HA gene of equine influenza virus, but there is no data about vaccine efficacy of these recombinants.